Year: 2005
Type: article
Abstract: Catalase-peroxidases (KatG) are bifunctional enzymes possessing both catalase and peroxidase activities. Three crystal structures of different KatGs revealed the presence of a novel Met-Tyr-Trp cross-link that has been suggested to impart catalatic activity to the KatGs. High-performance liquid chromatographic separation of the peptide fragments resulting from tryptic digestion of recombinant Mycobacterium tuberculosis WT KatG identified a peptide with unusual UV-visible spectroscopic features attributable to the Met(255)-Tyr(229)-Trp(107) cross-link, whose structure was confirmed by mass spectrometry. WT KatG lacking the Met-Tyr-Trp cross-link was prepared, making possible studies of its formation under oxidizing conditions that generate either compound I (peroxyacetic acid, PAA) or compound II (2-methyl-1-phenyl-2-propyl hydroperoxide, MPPH). Incubation of this "cross-link-free" WT KatG with PAA revealed complete formation of the Met-Tyr-Trp structure after six equivalents of peracid were added, whereas MPPH was unable to promote cross-link formation. A mechanism for Met-Tyr-Trp autocatalytic formation by KatG compound I is proposed from these studies. Optical stopped-flow studies of WT KatG and KatG(Y229F), a mutant in which the cross-link cannot be formed, were performed with MPPH and revealed an unusual compound II spectrum for WT KatG, best described as (P.)Fe(III), where P. represents a protein-based radical. This contrasts with the oxoferryl compound II spectrum observed for KatG(Y229F) under identical conditions. The structure-function-spectroscopy relationship in KatG is discussed with relevance to the role that the Met-Tyr-Trp cross-link plays in the catalase-peroxidase mechanism.
Source: Journal of Biological Chemistry
Institution: University of California, San Francisco
Language: English
Subfield: Inorganic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
Awards: G1257545902, G1988084045, G3729622492, G3891730877, G4623783088, PURCHASE OF Q TOF MASS SPECTROMETER, Bio-Organic Biomedical Mass Spectrometry Resource, Bio-Organic Biomedical Mass Spectrometry Resource, BIOCHEMISTRY AND HEPATIC TOXICITY OF CARBON RADICALS, Role of Mtb KatG in Isoniazid Activation and Resistance, Structure Biology and Targeted Drug Design for AIDS, G9000119760
OpenAlex ID: W2053145402